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AD_________________

Award Number: W81XWH-12-2-0014

TITLE: Development of Antibacterials Targeting the MEP Pathway of Select Agents

PRINCIPAL INVESTIGATOR: Robin Couch, PhD

CONTRACTING ORGANIZATION: George Mason University, Fairfax, VA 22030

REPORT DATE: March 2015

TYPE OF REPORT: Annual

PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012

DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited

The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.

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REPORT DOCUMENTATION PAGE Form Approved

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2. REPORT TYPEAnnual

3. DATES COVERED10 Feb 2014 - 9 Feb 2015

4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER

Development of Antibacterials Targeting the MEP Pathway of Select Agents 5b. GRANT NUMBERW81XWH-12-2-00145c. PROGRAM ELEMENT NUMBER

6. AUTHOR(S)Robin Couch

5d. PROJECT NUMBER

5e. TASK NUMBER

E-Mail: rcouch@gmu.edu 5f. WORK UNIT NUMBER

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORTNUMBER

George Mason University Ann T. McGuigan 4400 University Dr Fairfax VA 22030-4422

9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012

11. SPONSOR/MONITOR’S REPORTNUMBER(S)

12. DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited

13. SUPPLEMENTARY NOTES

14. ABSTRACTThe threat of bioterrorism and the use of biological weapons against both military personnel and civilian populations has become an increasing concern for governments around the world. The 1984 Rajneeshee Salmonella attack, 2001 anthrax letter attacks, 2003 SARS outbreak, 2009 H1N1 swine flu pandemic, and the current US flu epidemic all illustrate our vulnerability to both deliberate and natural outbreaks of infectious disease and underscore the necessity of effective antimicrobial and antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistance can be engineered into bacteria further highlights the need for continued development of novel antibiotics against new bacterial targets. This research project directly addresses this need through the development of a broad spectrum inhibitor of the biothreat agents Francisella tularensis and Yersinia pestis.

During this period of performance, we have utilized our optimized assays with the Y. pestis MEP synthase and the F. tularensis MEP cytidylyltransferase to screen molecular libraries and identify effective inhibitors of both MEP synthase and MEP cytidylyltransferase.

15. SUBJECT TERMS Enzymology, MEP pathway, bacteria, isoprene, drug discovery, antibiotics, screening, biothreat, inhibitors16. SECURITY CLASSIFICATION OF: 17. LIMITATION

OF ABSTRACT 18. NUMBEROF PAGES

19a. NAME OF RESPONSIBLE PERSONUSAMRMC

a. REPORTU

b. ABSTRACTU

c. THIS PAGEU UU

9 19b. TELEPHONE NUMBER (include area code)

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Table of Contents

Page

Introduction…………………………………………………………….………..…..4

Body…………………………………………………………………………………..4

Key Research Accomplishments………………………………………….……..8

Reportable Outcomes………………………………………………………………8

Conclusion……………………………………………………………………………8

References…………………………………………………………………………….8

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Introduction The long term objective of this research is to identify and develop a broad spectrum inhibitor of Francisella tularensis and Yersinia pestis. The methylerythritol phosphate (MEP) biosynthetic pathway of Francisella tularensis and Yersinia pestis provide multiple enzymes that may be targeted for inhibitor development. This pathway is utilized by bacteria, apicomplexan protozoa, and plants for isoprenoid biosynthesis. Isoprenic compounds are vital for cellular processes such as electron transport, cell wall and membrane biosynthesis, and signal transduction. Despite their structural and functional diversity, all isoprenoids are derived from two building blocks, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, which originate from either the MEP pathway or the mevalonic acid (MVA) pathway depending on the organism. Humans acquire isoprenes through the nonhomologous MVA pathway, making enzymes in the MEP pathway very attractive targets for antimicrobial development.

Body We hypothesize that inhibitors of the MEP pathway in Francisella tularensis and Yersinia pestis will serve as effective antibiotics by blocking isoprene biosynthesis. In strong support of this hypothesis, we have demonstrated the dose-dependent inhibition of F. tularensis and Y. pestis growth in vitro using the compounds fosmidomycin and FR900098 (Figure 1).

10-2 10-1 100 101 102 1030.0

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R2=0.95IC50 = 29.3 +/- 1.3 μM

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Figure 1. Growth inhibition of F. tularensis (top) and Y. pestis (bottom) by the compounds fosmidomycin and FR900098 (these molecules inhibit MEP synthase, an enzyme in the MEP pathway). The structures of the compounds are shown.

FR900098

Fosmidomycin

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To test this hypothesis, the Couch lab at George Mason University is collaborating with Walter Reed Army Institute of Research (WRAIR) in the screening of compound diversity libraries using enzyme-based assays for lead inhibitor discovery, evaluation of lead inhibitors in microbial growth assays, determining X-ray crystal structures of the MEP pathway enzymes MEP synthase and MEP cytidylyltransferase in complex with inhibitors, and using this information to design and synthesize novel broad spectrum antibacterials.

MEP Synthase (IspC) During this period of effort, we used a combination of affinity column chromatography,

mass spectrometry, and enzymology to definitively identify the active component in e29, the natural product inhibitor described in previous reports. A manuscript detailing this research is currently in preparation. As this inhibitor is the first of its kind, able to allosterically inhibit the enzyme, crystal screens with the Yersinia pestis MEP Synthase in complex with the inhibitor are presently underway.

As depicted in the Figure below, we have also performed a high-throughput screen of MEP Synthase using a commercially purchased molecular library (LOPAC library). Due to its wide dynamic range of activity, the primary screen was performed with the Y. pestis MEP Synthase. To ensure broad spectrum activity, a secondary screen was then performed with purified

Figure 2. Schematic of the approach to performing the high-throughput screen with IspC.

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recombinant Mycobacterium tuberculosis MEP Synthase and the 232 hits identified in the primary screen. To eliminate false positives (compounds that have innate absorption at 340 nm, thereby interfering with the NADPH signal (∆A340)), a tertiary screen was then performed, using an assay coupling MEP Synthase with MEP Cytidylyltransferase (IspD), as illustrated in Figure 3. The seven molecules demonstrating inhibition in both the single and coupled assays areconsidered hit compounds. Bacterial growth inhibition assays with these top seven compounds are currently underway. Screening of a second commercially available molecular library is also underway.

MEP Cytidylyltransferase (IspD) As illustrated in Figure 4, we have also performed a high-throughput screen with the

Francisella tularensis MEP Cytidylyltransferase. As the enzyme assay is coupled to the activity of pyrophosphatase, a tertiary screen was performed with the latter enzyme alone to assure the hit compounds are selective for IspD (Figure 5). Bacterial growth inhibition assays with these top five compounds are currently underway. Screening of a second commercially available molecular library is also underway.

Figure 3. Schematic of the approach to performing the tertiary screen with IspC.

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Figure 5. Schematic of the approach to performing the tertiary screen with pyrophosphatase and IspD.

Figure 4. Schematic of the approach to performing the high-throughput screen with IspD.

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Key Research Accomplishments

• Optimization of MEP synthase and MEP cytidylyltransferase assays for HTS.

• Screening of molecular libraries for inhibitors of MEP synthase and MEPcytidylyltransferase.

• Identification of a previously unknown allosteric site on the MEP synthase enzyme fromF. tularensis, Y. pestis, and M. tuberculosis.

• On-demand production and delivery of recombinant proteins to WRAIR for X-raycrystallography.

Reportable Outcomes

Haymond A, Johny C, Dowdy T, Schweibenz B, Villarroel K, Young R, Mantooth CJ, Patel T, Bases J, Jose GS, Jackson ER, Dowd CS, and Couch RD. Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase). PLoS One. 2014 Aug 29;9(8):e106243.

Chofor, R., Risseeuw, M.D., Pouyez, J., Johny, C., Wouters, J., Dowd, C.S., Couch, R.D., Van Calenbergh, S. Synthetic Fosmidomycin Analogues with Altered Chelating Moieties Do Not Inhibit 1-Deoxy-D-xylulose 5-phosphate Reductoisomerase or Plasmodium falciparum Growth In Vitro. Molecules. 2014; 19(2):2571-2587.

Jackson, E.R., San Jose, G., Brothers, R.C., Edelstein, E.K., Sheldon, Z., Haymond, A., Johny, C., Boshoff, H.I., Couch, R.D., and Dowd, C.S., The effect of chain length and unsaturation on Mtb Dxr inhibition and antitubercular killing activity of FR900098 analogs. Bioorganic & Medicinal Chemistry Letters, 2014 Jan 15;24(2):649-53.

• Funds for this project are used to support a lab technician (Ms. Chinchu Johny) and agraduate student (Ms. Amanda Haymond).

Conclusion

In summary, during this fiscal period, we have optimized assays with the Y. pestis MEP synthase and the F. tularensis MEP cytidylyltransferase for use in HTS. The screening of a commercially available library has identified several hit molecules for each of these enzymes. We have also identified the natural product allosteric inhibitor of MEP synthase.

During the next fiscal period, we will focus on screening a second large and chemically diverse molecular library for additional inhibitors of MEP synthase and MEP cytidylyltransferase.

References 1. Jawaid S, Seidle H, Zhou W, Abdirahman H, Abadeer M, et al. (2009) Kinetic

characterization and phosphoregulation of the Francisella tularensis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase). PLoS ONE 4: e8288. doi:10.1371/journal.pone.0008288.

2. Tsang A, Seidle H, Jawaid S, Zhou W, Smith C, et al. (2011) Francisella tularensis 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase: kinetic characterization andphosphoregulation. PLoS ONE 6: e20884. doi:10.1371/journal.pone.0020884.

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3. Zhang, Chung, Oldenburg (1999) A Simple Statistical Parameter for Use in Evaluation andValidation of High Throughput Screening Assays. J Biomol Screen 4: 67–73.

4. Henriksson LM, Unge T, Carlsson J, Aqvist J, Mowbray SL, et al. (2007) Structures ofMycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase providenew insights into catalysis. J Biol Chem 282: 19905–19916. doi:10.1074/jbc.M701935200.

5. Björkelid C, Bergfors T, Unge T, Mowbray SL, Jones TA (2012) Structural studies onMycobacterium tuberculosis DXR in complex with the antibiotic FR-900098. ActaCrystallographica Section D 68: 134–143. doi:10.1107/S0907444911052231.

6. San Jose G, Jackson ER, Uh E, Johny C, Haymond A, et al. (2013) Design of PotentialBisubstrate Inhibitors against Mycobacterium tuberculosis (Mtb) 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)-Evidence of a Novel Binding Mode. Medchemcomm 4:1099–1104. doi:10.1039/C3MD00085K.

7. Koppisch AT, Fox DT, Blagg BSJ, Poulter CD (2002) E. coli MEP synthase: steady-statekinetic analysis and substrate binding. Biochemistry 41: 236–243.